Neutron reflectometry (NR) and fluorescence spectroscopy were employed to uncover molecular details of the interaction between &#945;-syn and two anionic lipids, phosphatidic acid (PA) and phosphatidylserine (PS). NR data show that &#945;-syn comparably penetrates both PA- and PS-containing sparsely-tethered bilayer lipid membrane even though &#945;-syn binds more tightly to PA. Consistent penetration depths on the residue level were obtained using three single-Trp variants (F4W, Y39W, and F94W), and thus no correlation was found between binding affinity to and &#945;-syn penetration depth into either PA or PS membranes. The spectroscopic properties of W39 were found to be sensitive to the chemical nature of the membrane surface, with greater conformational heterogeneity in this region for PS-bound &#945;-syn. Our spectroscopic data also show that position 94 penetrates both PA and PS membranes comparably to position 4, though previous reports dictate that the former should be water-exposed while the latter is buried in the bilayer. Taken together, we suggest the N- and C-terminal regions near positions 4 and 94 are anchored to the membrane, while the putative linker spanning residue 39 samples multiple conformations, which are sensitive to the chemical nature of the membrane surface. This flexibility may enable &#945;-syn to bind diverse biomembranes in vivo. We have begun to study &#945;-syn clearance in the lysosome, a cellular site for proteolysis. To date, cathepsin D (CtsD) is the only lysosomal protease implicated in &#945;-syn proteolysis. However, we have found that CtsD does not completely proteolyze &#945;-syn and generates amyloid-forming truncated C-terminal species. Thus, the strong in vivo correlation between overexpression of CtsD and &#945;-syn levels as well as the neurotoxic potential of overexpressed &#945;-syn is puzzling. Other proteases and/or environmental factors must be needed to facilitate degradation and to avoid &#945;-syn aggregation in vivo. To address this, degradation of recombinant &#945;-syn by purified mouse brain and liver lysosomes as well as purified human cathepsins was characterized by peptide mapping using liquid chromatography mass spectrometry. The degradation process of &#945;-syn in lysosomal extracts has been completely mapped by mass spectrometry. Selective protease inhibition experiments with purified lysosomes established that cysteine cathepsins are involved in lysosomal degradation of recombinant &#945;-syn. Unlike CtsD which is an aspartyl protease, they degrade the central portion of &#945;-syn and circumvent protein aggregation. Using purified human proteases as references, peptide mapping showed that the cysteine cathepsin activity involve cathepsin B and L. Both enzymes cleave within the amyloid core region of &#945;-syn and inhibit aggregation. In order for CtsD to have improved efficacy, the presence of anionic lipids was required. This result is of biological relevance as intralysosomal vesicles may contain up to 30% of anionic BMP (bis(monooleoylglycero)phosphate) lipid; moreover, it could reconcile prior in vivo and in vitro results, as lipids were absent in the in vitro studies. Nevertheless, our data still indicate that CtsL is the most efficient in degrading &#945;-syn and suggest that cysteine cathepsins are essential. Individual cathepsin activities of CtsD, CtsB, and CtsL on &#945;-syn have been fully characterized in the presence of lipid vesicles. Remarkably, CtsL fully degrades &#945;-syn amyloid fibrils. The role of individual cathepsins has been revealed: both soluble and membrane-bound &#945;-syn is digested by both CtsB and CtsL whereas CtsD can only fully degrade the membrane-bound form, and aggregated &#945;-syn can only be dealt with by CtsL.